A5060113315- Enzyme Structure and Function
- Biochemical and Molecular Research
- Protein Structure and Dynamics
- Folate and B Vitamins Research
- RNA and protein synthesis mechanisms
- DNA and Nucleic Acid Chemistry
- Porphyrin Metabolism and Disorders
- HIV/AIDS drug development and treatment
- Metabolism and Genetic Disorders
- Bacterial Genetics and Biotechnology
- thermodynamics and calorimetric analyses
- Protein purification and stability
- Drug Transport and Resistance Mechanisms
- Pancreatic function and diabetes
- Diet, Metabolism, and Disease
- Amino Acid Enzymes and Metabolism
- Bacteriophages and microbial interactions
- Enzyme Catalysis and Immobilization
- Crystallography and molecular interactions
- Metabolism, Diabetes, and Cancer
- Molecular Sensors and Ion Detection
- Analytical Chemistry and Chromatography
- DNA Repair Mechanisms
- Protein Interaction Studies and Fluorescence Analysis
- Biochemical effects in animals
University of Tennessee at Knoxville
2014-2025
Knoxville College
2018-2019
Winston-Salem State University
2013
National Institute of Environmental Health Sciences
2003-2007
National Institutes of Health
2000-2007
Oak Ridge National Laboratory
2007
Harvard University
2005
Dana-Farber Cancer Institute
2005
Pennsylvania State University
2001
National Heart Lung and Blood Institute
2000
The crystal structures and enzymic properties of two mutant dihydrofolate reductases ( Escherichia coli ) were studied in order to clarify the functional role an invariant carboxylic acid (aspartic at position 27) substrate binding site. One mutation, constructed by oligonucleotide-directed mutagenesis, replaces Asp 27 with asparagine; other is a primary-site revertant Ser . only structural perturbations involve internally bound water molecules. Both mutants have low but readily measurable...
Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis cloned Escherichia coli gene. The mutations—at residue 27, aspartic acid replaced with asparagine; at 39, proline cysteine; and 95, glycine alanine—were designed to answer questions about relations between molecular structure function that raised x-ray crystal structures. Properties mutant proteins show Asp-27 is important for catalysis perturbation local a conserved cis peptide...
Protein structures are stabilized using noncovalent interactions. In addition to the traditional interactions, newer types of interactions thought be present in proteins. One such interaction, an anion-π pair, which positively charged edge aromatic ring interacts with anion, forming a favorable anion-quadrupole has been previously proposed [Jackson, M. R., et al. (2007) J. Phys. Chem. B111, 8242-8249]. To study role stabilizing protein structure, we analyzed pairwise between phenylalanine...
Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules their complexes, reporting on size shape in free solution. The purpose this multi-laboratory study was establish the precision accuracy basic data dimensions AUC validate previously proposed calibration techniques. Three kits cell assemblies containing radial temperature tools bovine serum albumin (BSA) reference sample were...
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAn engineered disulfide bond in dihydrofolate reductaseJesus E. Villafranca, Elizabeth Howell, Stuart J. Oatley, Nguyen Huu Xuong, and Joseph KrautCite this: Biochemistry 1987, 26, 8, 2182–2189Publication Date (Print):April 1, 1987Publication History Published online1 May 2002Published inissue 1 April 1987https://pubs.acs.org/doi/10.1021/bi00382a017https://doi.org/10.1021/bi00382a017research-articleACS PublicationsRequest reuse permissionsArticle...
Significance There is immense difficulty in mapping out the complete details of an enzyme’s mechanism, especially those that catalyze acid-base reaction, owing to simple fact hydrogen atom positions are rarely known with any confidence. Ultrahigh-resolution X-ray and, better still, neutron crystallography can provide this crucial layer information. We paired these techniques reveal catalytic mechanism dihydrofolate reductase (DHFR), enzyme necessary for nucleotide biosynthesis and a...
Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of binding reaction. In biological sciences, macromolecular interactions are essential in machinery cell. Experimental conditions, such as buffer and temperature, can be tailored to particular system being studied. However, careful planning needed since certain ligand macromolecule concentration ranges necessary obtain data. Concentrations need accurately determined reliable results....
Noncovalent interactions are quite important in biological structure-function relationships. To study the pairwise interaction of aromatic amino acids (phenylalanine, tyrosine, tryptophan) with anionic (aspartic and glutamic acids), small molecule mimics (benzene, phenol or indole interacting formate) were used at MP2 level theory. The overall energy associated an anion-quadrupole is substantial (-9.5 kcal/mol for a benzene-formate planar dimer van der Waals contact distance), indicating...
Dihydrofolate reductase from wild-type Escherichia coli (WT-ECDHFR) and a mutant enzyme in which aspartate 27 is replaced by asparagine have been compared with respect to the binding of inhibitor methotrexate (MTX). Although Asp27----Asn substitution causes only small changes association rate constants (kon) for formation binary ternary (with NADPH) complexes, dissociation these complexes (koff) are increased factors about 5- 100-fold, respectively, at pH 7.65. In experiments, initial MTX...
Hydrogen atoms play a central role in many biochemical processes yet are difficult to visualize by x-ray crystallography. Spallation neutron sources provide new arena for protein crystallography with TOF measurements enhancing data collection efficiency and allowing hydrogen be located smaller crystals of larger biological macromolecules. Here we report 2.2-Å resolution structure Escherichia coli dihydrofolate reductase (DHFR) complex methotrexate (MTX). Neutron were collected on 0.3-mm 3 D...
The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain accomplished using genetic biochemical criteria, including Southern analysis chromosomal DNA. mutation is stable E. K549 [thyA polA12 (Ts)] can be successfully transduced to other strains providing they have mutations their thymidylate synthetase (thyA) genes. A preliminary investigation relationship between fol thyA expression suggests...
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConstruction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role N-terminus in proteinLisa J. Reece, Robert Nichols, Richard C. Ogden, Elizabeth E. HowellCite this: Biochemistry 1991, 30, 45, 10895–10904Publication Date (Print):November 12, 1991Publication History Published online1 May 2002Published inissue 12 November...
Optimal enzyme activity depends on a number of factors, including structure and dynamics. The role is well recognized; however, the linkage between protein dynamics has given rise to contentious debate. We have developed an approach that uses aqueous mixture organic solvent control functionally relevant (without changing structure), which in turn modulates activity. Using this approach, we predicted hydride transfer reaction catalyzed by dihydrofolate reductase (DHFR) from Escherichia coli...
Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) catalyzes the same reaction as chromosomal counterpart but is highly resistant to widely used antibiotic Trimethoprim (TMP) unlike enzyme. The structure of Q67H mutant DHFR complexed with a non-specific inhibitor Congo red (CGR) has been determined at 1.15 Å resolution. In Fo-Fc map, one two naphthalene moieties in CGR clearly observed, however, biphenyl linker and other moiety are not seen owing flexibility. does utilize its...
The apoenzyme of wild-type (WT) dihydrofolate reductase (DHRF) from Escherichia coli exists in two conformational states, Et and Ew, which differ affinity for NADPH kinetic competence. Dissociation constants the binary complex with conformers by over 100-fold (KDt = 0.17 microM, KDw 22 microM). Rate governing interconversion are small (t1/2 Ew----Et 71 s), since Ew is not catalytically competent, this conversion accompanied an increase catalytic velocity. equilibrium proportion absence...
Type II dihydrofolate reductase (DHFR) is a plasmid-encoded enzyme that confers resistance to bacterial DHFR-targeted antifolate drugs. It forms symmetric homotetramer with central pore which functions as the active site. Its unusual structure, results in promiscuous binding surface accommodates either (DHF) substrate or NADPH cofactor, has constituted significant limitation efforts understand its specificity and reaction mechanism. We describe here first structure of ternary R67...
Abstract The statistical analysis of aromatic rings program allows for an automated search anion‐π interactions between phenylalanine residues and carboxylic acid moieties neighboring aspartic or glutamic in protein data bank (PDB) structures. is written C++ available both as a standalone code through web implementation that users to upload analyze biomolecular structures PDB format. outputs lists Phe/Glu Phe/Asp pairs involved potential interactions, together with geometrical (distance...
R67 dihydrofolate reductase (DHFR) is an R-plasmid-encoded enzyme that confers resistance to the antibacterial drug, trimethoprim. This DHFR variant not homologous in either sequence or structure chromosomal DHFRs. A recent crystal of active tetrameric species describes a single site pore traverses length protein (Narayana et al., 1995). Related sites (due 222 symmetry element at center pore) are used for binding ligands, i.e., each half-pore can accommodate substrate, dihydrofolate,...
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConstruction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductaseElizabeth Ehrhardt Howell, Mark S. Warren, Carol L. J. Booth, Jesus E. Villafranca, and Joseph KrautCite this: Biochemistry 1987, 26, 8591–8598Publication Date (Print):December 1, 1987Publication History Published online1 May 2002Published inissue 1 December...
Metformin is the most commonly prescribed treatment for type II diabetes and related disorders; however, molecular insights into its mode(s) of action have been limited by an absence structural data. Structural considerations along with a growing body literature demonstrating effects on one-carbon metabolism suggest possibility folate mimicry anti-folate activity. Motivated recognition that anti-diabetic biguanides may act directly upon gut microbiome, we determined structures complexes...
Plasmid-encoded R67 dihydrofolate reductase (DHFR) catalyzes a hydride transfer reaction between substrate (DHF) and its cofactor, nicotinamide adenine dinucleotide phosphate (NADPH). DHFR is homotetramer that exhibits numerous characteristics of primitive enzyme, including promiscuity in binding formation nonproductive complexes, the absence conserved acid active site. Furthermore, R67's site pore, which mostly accessible by bulk solvent. This study uses computational approach to...
R67 dihydrofolate reductase (DHFR) is a type II DHFR produced by bacteria as resistance mechanism to the increased clinical use of antibacterial drug trimethoprim. Type DHFRs are not homologous in either sequence or structure with chromosomal DHFRs. The enzymes contain four identical subunits which form homotetramer containing single active site pore accessible from end. Although crystal complex folate has been reported [Narayana et al. (1995) Nat. Struct. Biol. 2, 1018], nature ternary must...